Visualizing Metal Content and Intracellular Distribution in Primary Hippocampal Neurons with Synchrotron X-Ray Fluorescence

Abstract

Increasing evidence suggests that metal dyshomeostasis plays an important role in human neurodegenerative diseases. Although distinctive metal distributions are described for mature hippocampus and cortex, much less is known about metal levels and intracellular distribution in individual hippocampal neuronal somata. To solve this problem, we conducted quantitative metal analyses utilizing synchrotron radiation X-Ray fluorescence on frozen hydrated primary cultured neurons derived from rat embryonic cortex (CTX) and two regions of the hippocampus: dentate gyrus (DG) and CA1. Comparing average metal contents showed that the most abundant metals were calcium, iron, and zinc, whereas metals such as copper and manganese were less than 10% of zinc. Average metal contents were generally similar when compared across neurons cultured from CTX, DG, and CA1, except for manganese that was larger in CA1. However, each metal showed a characteristic spatial distribution in individual neuronal somata. Zinc was uniformly distributed throughout the cytosol, with no evidence for the existence of previously identified zinc-enriched organelles, zincosomes. Calcium showed a peri-nuclear distribution consistent with accumulation in endoplasmic reticulum and/or mitochondria. Iron showed 2–3 distinct highly concentrated puncta only in peri-nuclear locations. Notwithstanding the small sample size, these analyses demonstrate that primary cultured neurons show characteristic metal signatures. The iron puncta probably represent iron-accumulating organelles, siderosomes. Thus, the metal distributions observed in mature brain structures are likely the result of both intrinsic neuronal factors that control cellular metal content and extrinsic factors related to the synaptic organization, function, and contacts formed and maintained in each region.

Biogenesis of zinc storage granules inDrosophila melanogaster

“…Upon feeding on a diet with high content of zinc, C. elegans generates new granules within intestinal cells to store the excess metal. Thus zinc storage granules, or zincosomes as they have been also called (Beyersmann and Haase, 2001;Colvin et al, 2016), appear to be a conserved LRO in animal biology. Insect zinc storage granules were first described in M. domestica (Sohal et al, 1976), D. hydei (Zierold and Wessing, 1990) and have also been observed in termites (Stewart et al, 2011).…”

Elemental characterisation of the pyramidal neuron layer within the rat and mouse hippocampus

“…14,[52][53][54][55][56][57] For these reasons, XFI has found increasing use within the field of neuroscience, to study elemental alterations that correlate with anatomical structure, 58,59 or occur as a consequence of diseases such as Alzheimer's disease, [60][61][62][63][64] Parkinson's disease, 65,66 damaging events that occur during epilepsy, [67][68][69] traumatic brain injury, 70 and stroke. 17,71,72 XFI has also been used to study the elemental phenotype of individual cells and neurons within in vitro cultures, 64,[73][74][75][76] and to study the elemental distribution of the rat hippocampus, 12,[67][68][69]77 but detailed characterisation of elemental signatures of cells within CA1 medial, CA1 lateral, CA2 or CA3 sub-regions has not been previously undertaken.…”

X-ray Fluorescence Nanotomography of Single Bacteria with a Sub-15 nm Beam

“…Trace elements are essential for carrying out biochemical reactions and act as structural components in cells. The appreciation of the important roles that trace elements, especially metals, play in cell metabolism has resulted in an increase in the number of studies in this area 1 – 5 . However, to truly understand the functions of trace elements in cells and tissues, the distribution within a cell must be imaged and quantified in the native cellular environment, which has proven to be difficult.…”